PROJECT SUMMARY/ABSTRACT Chimeric-antigen receptor (CAR) T cells have opened a new era for targeted immunotherapy of cancer. The applicant's mentor, Dr. Carl June, is recognized as a world leader in the field of genetically-engineered T cell therapy for hematologic malignancy. Although anti-CD19 CAR T cells (CART19) generate unprecedented responses in patients with B-cell leukemia and lymphoma, relapse represents the major cause of treatment failure. The long term goal of this proposal is to develop an independent research career focused on improving targeted immunotherapy by studying and neutralizing tumor evasion mechanisms. The unifying objective of this application is to design novel strategies for the treatment and prevention of tumor immunoescape based on the mechanisms leading to relapse, using CART19 as a model. The central hypothesis is that multiple mechanisms can lead to escape from immune attack; therefore, combined approaches targeting the problem from multiple sides will prevent escape. The rationale for this research is that identification of key tumor escape mechanisms would allow us to develop specific remedies for these. The central hypothesis of this proposal will be tested by pursuing two specific aims: 1) to characterize the pathogenesis of leukemia relapses occurring after CD19-targeted immunotherapies by studying minor CD19-negative resistant clones present before CART19 treatment and by identifying key resistance pathways in CD19-positive relapses using functional genomics; 2) To design dual-specific chimeric antigen receptor T cells that will prevent tumor escape. This research will be significant because it will contribute depth (of understanding the mechanisms of relapse) and breadth (of novel potentially curative therapy) to the immunotherapeutic arsenal against cancer. Ultimately, such knowledge has the potential to vertically advance the field of CAR-redirected T cell immunotherapy as well as other targeted immunotherapies. This is project is innovative because it combines high throughput screening techniques together with in vitro and in vivo functional studies to study the possible mechanisms of resistance after immunotherapy and therefore to generate novel treatments. The proposed research activities are crucial to the development of the applicant as an independently-funded scientist with a focus on cellular immunotherapy. Dr. Ruella will receive further training in molecular biology, translational medicine, and immunology from his mentors and training in next-generation sequencing, cancer biology and bioinformatics from experienced collaborators at the University of Pennsylvania. Therefore at the conclusion of the training period, the applicant will have acquired a unique set of intellectual and technical skills that will allow him to attack the problem of resistance to targeted immunotherapies from several angles at once. In addition, this award will support a unique training experience in translational research and will establish an academic pathway for the discovery and development of new CAR T cell approaches.